Bispecific soluble cytokine receptor-nanobody fusions inhibit Interleukin (IL-)6 trans-signaling and IL-12/23 or tumor necrosis factor (TNF) signaling

At least 0.5% of people in the Western world develop inflammatory bowel disease (IBD). While antibodies that block tumor necrosis factor (TNF) α and Interleukin (IL-)23 have been approved for the treatment of IBD, IL-6 antibodies failed in the phase II clinical trial due to non-tolerable side effects. However, two clinical phase II studies suggest that inhibiting IL-6/soluble IL-6R (sIL-6R)-induced trans-signaling via the cytokine receptor gp130 benefit IBD patients with fewer adverse events. Here we develop inhibitors targeting a combination of IL-6/sIL-6R and TNF or IL-12/IL-23 signaling, named cs130-TNFVHHFc and cs130-IL-12/23VHHFc. Surface plasmon resonance experiments showed that recombinant cs130-TNFVHHFc and cs130-IL-12/23VHHFc bind with high affinity to IL-6/sIL-6R complexes and human TNFα (hTNFα) or IL-12/IL-23, respectively. Immunoprecipitation experiments have verified the higher ordered complex formation of the inhibitors with IL-6/sIL-6R and IL-12. We demonstrated that cs130-TNFVHHFc and cs130-IL-12/23VHHFc block IL-6/sIL-6R trans-signaling-induced proliferation and STAT3 phosphorylation of Ba/F3-gp130 cells, as well as hTNFα- or IL-23-induced signaling, respectively. In conclusion, cs130-TNFVHHFc and cs130-IL-12/23VHHFc represent a class of dimeric and bispecific chimeric cytokine inhibitors that consist of a soluble cytokine receptor fused to anti-cytokine nanobodies.

The sIL-6R-driven IL-6 trans-signaling has been mainly assigned as the pathological mode of IL-6 signaling that contributes to inflammatory, autoimmune diseases and cancer.In many disease models, soluble variants of gp130 (9) were shown to inhibit IL-6 trans-signaling specifically.Treatment with sgp130Fc, the dimeric IgG1-Fc fusion protein consisting of all six extracellular domains of gp130, was shown to be more beneficial compared to global blockade of classic and transsignaling by neutralizing antibodies including sepsis (10), cerulein-induced acute pancreatitis (11), bone fracture healing (12,13), and myocardial infarction (14).sgp130Fc was named Olamkicept for clinical development by the WHO in 2016.
At least 0.5% of people in the Western world develop inflammatory bowel disease (IBD) with symptoms like intestinal fibrosis, abscesses, and eventually colitis-related tumors (15).Antibodies blocking TNFα and IL-23 were approved for IBD (16).In contrast, IL-6 antibodies failed in phase II clinical trials for IBD due to non-tolerable side effects (17,18), including intestinal perforations which were also observed for anti-IL-6R therapy for rheumatoid arthritis (19).Of note, Olamkicept has recently passed two phase II clinical studies for IBD with promising results (20)(21)(22).
We have generated cs130, a size-reduced, highly active, and selective trans-signaling inhibitor (23).cs130 consists only of the first three extracellular cytokine binding domains D1-D3 of sgp130 fused to the a non-neutralizing nanobody VHH6 that binds to IL-6:sIL-6R complexes (23)(24)(25).Based on cs130, c19s130 was the first example of a bispecific trans-signaling inhibitor, which consists of cs130 plus the neutralizing nanobody VHH72 (26) directed against the S-RBD of SARS-CoV2 enabling the inhibition of IL-6 trans-signaling and cellular entry of SARS-CoV2 (27).
Here we refined cs130, resulting in a class of dimeric and bispecific chimeric cytokine inhibitors, which simultaneously block IL-6 trans-signaling and TNFα-or IL-12/23-signaling via fusion to nanobodies directed against TNFα (28,29), and the p40 subunit of IL-12 and IL-23 (30).Such bispecific inhibitors of centrally involved cytokines in IBD might be of therapeutic value for future therapeutic applications, especially once sgp130Fc has been approved for IBD.

cs130-TNF VHH Fc efficiently inhibits hTNFα-induced apoptosis
Next, we determined and compared the affinity of cs130-TNF VHH Fc, TNF VHH Fc, and recombinant human soluble TNFRIFc fusion protein (hsTNFRIFc) (32) to hTNFα by SPR (Fig. 4, A-D and Table 1).cs130-TNF VHH Fc, and TNF VHH Fc displayed comparable affinities of 149.9, and 159.8 PM for hTNFα, respectively, demonstrating that fusion to cs130 did not disturb the interaction of the second nanobody with hTNFα.Next, we performed competition experiments with hTNFα bound to hsTNFRIFc with increasing concentrations of the inhibitors by SPR (Fig. 4E).hTNFα bound to hsTNFRIFc with 92.49PM (Fig. 4D and Table 1), which agrees with the previously described affinity of 49 PM (33).12.5 nM of cs130-TNF VHH Fc, and TNF VHH Fc, whereas 6.25 nM of Etanercept (soluble TNFRIIFc, Enbrel) abrogated the binding of hTNFα to immobilized hsTNFRIFc (Fig. 4, E-H).
Due to the lack of TNF receptors, Ba/F3 cells did not respond to hTNFα.hTNFα can induce cellular apoptosis of L929 via activation of TNF receptor I (TNFRI) (34).To demonstrate the inhibition of hTNFα, we chose the L929 cell line which is commonly used for cell death assays.We treated L929 cells with 1 ng/ml hTNFα to determine the inhibitory capacity of cs130-TNF VHH Fc, TNF VHH Fc, and Etanercept.As depicted in Figure 5A, cs130-TNF VHH Fc, TNF VHH Fc, and Etanercept inhibited hTNFα-induced cell death of L929 in a dose-dependent manner.Notably, the inhibitory profile was comparable with IC 50 s of 0.28 nM, and 0.56 nM for cs130-TNF VHH Fc and TNF VHH Fc, respectively, whereas Etanercept was significantly better with an IC 50 of 3.13 pM.Of note, c19s130Fc did not prevent hTNFα-induced cell death of L929 (Fig. 5A).Apoptosis of L929 cells was quantified using flow cytometry after 24 h stimulation with 1 ng/ml hTNFα and 1, 10, and 100 nM cs130-TNF VHH Fc (Fig. 5B).Ethanol treatment served as a positive control for apoptosis, whereas TNFα-nontreated cells served as proliferation control.L929 cells were apoptotic after hTNFα stimulation (81%), whereas inhibition with cs130-TNF VHH Fc efficiently prevented apoptosis.About 82 to 84% of cells were still alive when treated with 10 or 100 nM cs130-TNF VHH Fc, respectively, but also the lowest concentration of 1 nM cs130-TNF VHH Fc showed 64% cell viability (Fig. 5B).L929 cells were stimulated with 0.1 ng/ml hTNFα and c19s-130Fc, cs130-TNF VHH    recombinant cs130-IL-12/23 VHH Fc, and recombinant HIL-6 with FLAG-beads.The precipitated bound fraction and the non-precipitated unbound fraction were analyzed by Western blotting (Fig. 7C, last two lanes).Western blotting revealed that FLAG-tagged HIL-12 was precipitated with FLAG-beads, which resulted in co-immunoprecipitation of cs130-IL-12/ 23 VHH Fc and HIL-6 (Fig. 7C, last two lanes).cs130-IL-12/ 23 VHH Fc, HIL-6 and HIL-12 were also incubated separately with FLAG-beads as control.Here, only FLAG-tagged HIL-12 was detected in the bound fraction by Western blotting, whereas cs130-IL-12/23 VHH Fc and HIL-6 remained in the unbound fraction (Fig. 7C, lanes 4-9).Lanes 1 to 3 of Figure 7C served as loading control using recombinant proteins.Our data demonstrated that cs130-IL-12/23 VHH Fc simultaneous inhibited IL-6 trans-signaling and IL-23 signaling which is mediated by the formation of a ternary 2xcytokine:cytokine inhibitor complex.

Discussion
Here, we describe the development of two dimeric and bispecific biomolecules consisting of a basic IL-6 transsignaling inhibitory module fused to inhibitory nanobodies targeting either hTNFα or IL-12/IL-23 signaling.The basic IL-6 trans-signaling module cs130 is made of the first three extracellular domains D1 to D3 of gp130, which binds IL-6:sIL-6R complexes.Of note, these domains alone facilitate only low-affinity binding (35).High-affinity binding in cs130Fc is achieved by fusion with VHH6, a non-neutralizing IL-6:sIL-6R selective nanobody (23).In contrast to sgp130, cs130 did not inhibit IL-11 trans-signaling (23).Bispecificity toward hTNFα, and IL-12/IL-23 was achieved by the fusion of an additional antagonistic nanobody directed against hTNFα or the shared p40 subunit of IL-12/IL-23 (29,30), which are located between the IL-6 trans-signaling inhibitor and the Fc part of an IgG1 antibody.The Fc part served as a dimerizer and eased the purification of the two novel cs130Fc variants, cs130-IL-12/23 VHH Fc and cs130-TNF VHH Fc.
Initially, bispecific antibodies (bsAbs), which bind two independent epitopes on the same or different antigens, were developed for therapeutic applications (36).Prominent examples are bsAbs as selective T cell engagers and activators with a binding site for a tumor-associated antigen and CD3 T cell co-receptor.Currently, more than 100 anti-cancer bsAbs are in clinical development, that prevent bacterial and viral infections, interfere with ligand/receptor interaction, mediate intracellular drug delivery, or result in serum half-life extension (37).The bispecific cs130 fusion proteins were highly effective in target binding due to neutralizing IL-6 transsignaling, TNF-induced apoptosis, and IL-12/IL-23 signaling.We also showed that the cs130-TNF VHH -Fc blocks the target cytokines simultaneously without influencing the individual domains, which would reduce the binding affinity.As mentioned above, sgp130 and variants are potent IL-6 trans-signaling inhibitors largely without affecting classic signaling (9).It seems that sgp130 also blocks inflammatory trans-presentation (cluster signaling), albeit results are still heterogeneous, and blocking might be context-dependent (8,38).Therapeutic targeting of IL-6 in chronic inflammatory diseases mainly relies on the two IL-6R antibodies tocilizumab and sarilumab, while the siltuximab directed against IL-6 has been approved for Castleman's disease (39).However, Tocilizumab has failed in phase II clinical trials for IBD due to nontolerable side effects (17,18), including intestinal perforations that were also occasionally observed in patients during anti-IL-6R therapy of rheumatoid arthritis (19).A recent publication described the development of bispecific nanobodies targeting IL-6 and TNF with additive efficacy in translational models of rheumatoid arthritis by inhibition of classic and trans-signaling of IL-6 and TNF signaling (40), a strategy that most likely will not work in IBD.The first small open-label phase IIa clinical trial with sgp130Fc (Olamkicept) included 16 patients with IBD (EudraCT No 2016-000205-36).Olamkicept was well tolerated and induced a clinical response in 44% and clinical remission in 19% of the patients (41).The second double-blind placebo-controlled phase IIb clinical study with 91 patients focused on moderate to severe ulcerative colitis (21).Clinical remission was not seen in the placebo group but in 6.7% of the patients receiving the lower dose of Olamkicept (300 mg/injection) and 20.7% receiving the higher dose of Olamkicept (600 mg/injection).Of note, mucosal healing was seen in 3.4% (placebo), 10% (Olamkicept 300 mg/injection), and 34.5% (Olamkicept 600 mg/injection) of the patients (20).Phase III trials are currently in preparation (22).One motivation to generate bispecific cs130Fc variants was based on these recent data suggesting that selective inhibition of trans-signaling might be superior over simultaneous blocking of classic and trans-signaling by IL-6R antibodies in IBD.
We selected nanobodies blocking the activity of hTNFα and IL-12/IL-23 because conventional IgG1 antibodies blocking TNFα and Interleukin (IL-)23 have been approved for the treatment of IBD ( 16), and therefore these targets are promising candidates to generate small-sized cs130 fusion proteins.Anti-TNFα agents, including Adalimumab, Infliximab, Certolizumab, and Golimumab are now used as biological goldstandard therapy for both colitis ulcerosa and Crohn's disease management (42,43).The TNF nanobody used in this study is part of Ozoralizumab, a trivalent humanized antibody consisting of two different TNFα nanobodies and a human serum albumin nanobody to increase serum half-life (44).Ozoralizumab was well tolerated and showed efficacy in two recent phase III clinical studies for rheumatoid arthritis (OHZORA and NATSURA trial) (45,46).The IgG1 antibody Ustekinumab, which targets IL-12/IL-23_p40 subunit, was recently approved for Crohn's disease (47).Like Ustekinumab, the IL-12/IL-23 nanobody used in this study also binds to p40 and prevents interaction of IL-12 and IL-23 with the common IL-12Rβ1 (30).
Taken together, the cs130Fc variants cs130-TNF VHH Fc and cs130-IL-12/23 VHH Fc are the first bispecific cytokine inhibitor fusion protein designs consisting of a soluble cytokine receptor and cytokine-targeting nanobodies.

Proliferation assays
Ba/F3-gp130 cells were washed three times and 5000 cells were cultured for 3 days in a final volume of 100 μl in the presence of cytokines and inhibitors.The CellTiter-Blue Reagent was used to determine cellular viability by recording the fluorescence (excitation 560 nm, emission 590 nm) using an Infinite M200 PRO plate reader (Tecan, Crailsheim, Germany) immediately after adding 20 μl of reagent per well (time point 0) and up to 120 min thereafter.
Cytokine stimulation of cells and lysate preparation 10 6 Ba/F3-gp130 or NK-92 cells/ml were washed and starved in serum-free medium for 5 h.L929 cells were seeded at a density of 5 × 10 5 cells per 60 mm dish 24 h prior stimulation and also washed five times with PBS before starving in serum-free DMEM for at least 5 h.Prior to stimulation, cytokines and inhibitors were pre-incubated at room temperature for 30 min.Subsequently, cells were stimulated with the indicated cytokines and inhibitor combinations for 20 min, harvested by centrifugation at 4 C for 1 min at 1500g, frozen and lysed.Protein concentration of cell lysates was determined by the BCA Protein Assay (Pierce, Thermo Scientific).Analysis of STAT3 activation was performed by Western blotting of 50 μg of total protein from total cell lysates and subsequent detection steps using the anti-pSTAT3 (Tyr705) (1:1000), anti-STAT3 (1:1000), γ-Tubulin (1:1000), and Caspase 3 (1:1000) antibodies described above.

Western blotting
Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane.Membranes were blocked and probed with the indicated primary antibodies.After washing, membranes were incubated with secondary peroxidase-conjugated antibodies (1:2500) or fluorescencelabeled secondary antibodies (1:10,000).The Immobilon Western Reagents (Millipore Corporation) and the Chemo-Cam Imager (INTAS Science Imaging Instruments GmbH) or the Odyssey Fc Imaging System (LI-CORE Biosciences) were used for signal detection.
Expression and purification of cs130-TNF VHH Fc and cs130-IL12/IL-23 VHH Fc Mammalian expression plasmids encoding cs130-TNF VHH Fc and cs130-IL-23 VHH Fc were transfected into Expi-293F cells using ExpiFectamine.Reaching 4.5 to 5.5 × 10 6 c/ml, the cells were diluted to a final density of 3 × 10 6 c/ml in 30 ml Expi293F expression medium for transfection.30 μg of the plasmid expression vectors were used for transfection.Henceforth, the culture was harvested by centrifugation at 450g at 4 C for 5 min, followed by centrifugation of the resulting supernatant at 4000g at 4 C for 20 min.The supernatant of the second centrifugation step was filtered (0.45 μm, Carl Roth cat.#P667.1)and purified by affinity chromatography.Supernatant was loaded on a ProteinA column (1 ml HiTrap MabSelect PrismA; GE Healthcare) at a flow rate of 1 ml/min.The column was then washed with 30 column volumes of PBS.Proteins were eluted at pH 3.2 to 3.5 using a 50 mM citric acid buffer.Fractions containing the protein peak were pooled, and the pH was adjusted to pH 7 with 1 M Tris.Proteins were buffer exchanged to PBS using illustra NAP25 (GE Healthcare Life Sciences) columns.Protein concentration was determined by measuring absorbance at 280 nm, and samples were flash-frozen in liquid nitrogen.2.5 μg of protein were loaded per lane and separated by SDS-PAGE under reducing (106 mM β-Mercaptoethanol, 95 C for 10 min) and non-reducing (without β-Mercaptoethanol and cooking) conditions.The gel was stained with Coomassie staining solution (80% ethanol, 20% acetic acid, 4% Coomassie brilliant blue R250) for 1 h and was destained overnight in destaining solution (20% ethanol, 10% acetic acid).

Surface plasmon resonance
For surface plasmon resonance experiments, the Biacore X100 instrument (GE Healthcare Life Sciences) was used.cs130-TNF VHH Fc, cs130-IL12/23 VHH Fc, TNF VHH Fc, and IL12/ IL-23 VHH Fc were captured to a single flow cell of a ProteinA sensorchip to reach 100 response units (RUs) of the analyte at maximal concentration.Three samples containing only running buffer were injected over both ligand and reference flow cell, followed by HIL-6, HIL-12 serially diluted from 50 to 0.1 nM, with a replicate of the 12.5 nM concentration.The analyte was injected at a flow rate of 30 μl/min for 120 s, and the dissociation was measured for 300 s. hsTNFRIFc (R&D Systems, #372-RI-050/CF) was immobilized in 10 mM acetate buffer (pH 5.5) by amine coupling on a CM5 chip (490 RU).After immobilization, hTNFα was injected at a flow rate of 30 μl/min at increasing concentrations (0.2-200 nM).Association was monitored in periods of 120 s, and the dissociation was measured for 400 s.Immobilized hsTNFRIFc was regenerated with 2 M MgCl 2 to remove bound hTNFα for multiple cycle measurement.25 nM hTNFα was injected in the presence of increasing concentrations of cs130-TNF VHH Fc, TNF VHH Fc, and Etanercept (0.2-25 nM).Experiments were carried out at 25 C in PBS pH 7.4, composed of 137 mM NaCl, 2.7 mM KCl, 12 mM HPO 4 2− und H 2 PO 4 − , and 0.05% (v/v) surfactant P20 (GE Healthcare).The resulting data were reference subtracted and fit to a 1:1 binding model using the Biacore X100 Evaluation software V 2.0.1.

L929-cytotoxicity assays with hTNFα
L929 cells were seeded on a 96-well plate with a density of 30.000 cells/well and cultured for 24 h.Next, the cells were incubated for 30 min at 37 C with 2.5 μg/ml Actinomycin D (Thermo Fisher Scientific, cat.#15452969).Afterwards, 1 ng/ ml hTNFα (Thermo Fisher Scientific, cat.# PHC3011) was added with or without indicated concentrations of cs130-TNF VHH Fc, TNF VHH Fc, and Etanercept and cultured for 24 h at 37 C.The cell viability was assessed with Calcein AM (Invitrogen, Thermo Fisher Scientific) following the manufacturer's instructions on an Infinite M200 PRO plate reader.
AnnexinV/7-AAD staining 1.25 × 10 5 L929 cells were used per 6 well and incubated with 1 ng/ml hTNFα with or without indicated concentrations of cs130-TNF VHH Fc for 24 h.Cells were washed twice with ice-cold PBS, if indicated with 70% ethanol, and resuspended in 300 μl Annexin V binding buffer (BD Bioscience) with 0.5 μl Annexin V-PE (ImmunoTools) for 15 min in the dark at RT. 1 μl 7-AAD (R&D Systems) was added before analysis via flow cytometry recording 20,000 events.

Immunoprecipitation pulldown assay
Anti-FLAG M2 affinity gel (Sigma Aldrich, FLAG-beads) was washed twice with TBS-Tween (0.05%) at 2700g for 2 min.HIL-6, HIL-12, and cs130-IL-12/23 VHH Fc were adjusted to a concentration of 10 μg/ml, and 15 μg/ml, respectively, in TBS-Tween (0.05%).Proteins were added separately as negative control to 30 μl ANTI-FLAG M2 affinity gel or mixed together in 1 M ratio for pulldown and incubated overnight at 4 C under gentle agitation.After centrifugation, an unbound fraction was collected, samples were washed twice with TBS-Tween (0,05%), and proteins were eluted with 100 μl 2.5× Laemmli buffer at 95 C for 10 min 20 μl of supernatant with 300 ng protein was subjected to Western blot analysis.

Figure 2 .
Figure 2. Binding of cs130-TNF VHH Fc and cs130-IL-12/23 VHH Fc to HIL-6.A, schematic illustration of surface plasmon resonance experiments.B, SPR analysis of c19s130Fc binding to HIL-6.C, SPR analysis of cs130-TNF VHH Fc binding to HIL-6.D, SPR analysis of cs130-IL-12/23 VHH Fc binding to HIL-6.(B-D) c19s130Fc, cs130-TNF VHH Fc, and cs130-IL-12/23 VHH Fc were immobilized on a Protein A chip and increasing concentrations of HIL-6 were injected.Sensorgrams in response units (RU) over time are depicted as colored lines, and global fit data are displayed as black lines.

Figure 5 .Figure 6 .
Figure 5. cs130-TNF VHH Fc blocks hTNFα induced apoptosis of L929 cells.A, L929 cells were stimulated 1 ng/ml hTNFα and Actinomycin D in the presence of increasing concentrations of c19s130Fc, cs130-TNF VHH Fc, TNF VHH Fc, and Etanercept.24 h post-stimulation, cellular proliferation was detected using the Calcein-assay.Assays are representative of three independent experiments.B, L929 cells were incubated for 24 h with 1 ng/ml hTNFα with indicated concentrations of cs130-TNF VHH Fc.Controls cells were either untreated or washed with 70% EtOH before the measurement for the EtOH condition.Analysis of cells stained with AnnexinV and 7-AAD by flow cytometry.The mean ±SD of three independent experiments was plotted in the graphs (* p < 0,05; **p < 0,01; *** p < 0,001; **** p < 0,001).C, cleaved Caspase3 in L929 cells treated with 0.1 ng/ml hTNFα in the presence of indicated concentrations of c19s130Fc, cs130-TNF VHH Fc, and TNF VHH Fc, hTNFα (+) or left untreated (−) for 12 h.Equal amounts of proteins (50 μg/lane) were analyzed via specific antibody detecting cleaved Caspase3 and total (uncleaved) Caspase3.Western blotting data show one representative experiment out of three.

Table 1
Surface plasmon resonance analysis of the mono-and bi-specific inhibitors against HIL-6, TNF, and HIL-12